Extract, consumable product and method for enriching bioactive metabolite in an extract

ABSTRACT

This disclosure relates to methods and compositions with enhanced levels of one or more tyramine containing hydroxycinnamic acid amides. Also disclosed herein are methods for producing a consumable product with enhanced levels of a tyramine containing hydroxycinnamic acid amide. Some embodiments relate to a composition enriched with a tyramine containing hydroxycinnamic acid.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/525,573, filed Nov. 12, 2021, issued May 16, 2023, as U.S. Pat. No.11,647,776, which is a continuation of PCT/US2020/059726 filed Nov. 9,2020, which claims the priority benefit of U.S. Provisional ApplicationNo. 62/933,660, filed Nov. 11, 2019, the entire content of each of whichis hereby incorporated by reference herein in its entirety.

BACKGROUND

N-Hydroxycinnamic acid amides (HCAAs) are synthesized by thecondensation of hydroxycinnamoyl-CoA thioesters and aromatic amines. Thehydroxycinnamoyl-CoA thioesters include cinnamoyl-CoA, p-coumaroyl-CoA,caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA, and are synthesized fromcinnamic acid by a series of enzymes, including cinnamate-4-hydroxylase,coumarate-3-hydroxylase, caffeic acid O-methyltransferase,ferulate-5-hydroxylase, and hydroxycinnamate:CoA ligase (Douglas (1996)Trends Plant Sci 1:171-178).

Tyramine-derived HCAAs are commonly associated with the cell wall oftissues near pathogen-infected or wound healing regions. Moreover,feruloyltyramine and feruloyloctapamine are covalent cell wallconstituents of both natural and wound periderms of potato (Solanumtuberosum) tubers, and are putative components of the aromatic domain ofsuberin. The deposition of HCAAs is thought to create a barrier againstpathogens by reducing cell wall digestibility. HCAAs are formed by thecondensation of hydroxycinnamoyl-CoA thioesters with phenylethylaminessuch as tyramine, or polyamines such as putrescine. The ultimate step intyramine-derived HCAA biosynthesis is catalyzed byhydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl) transferase. In viewof the important role of these compounds, it is desirable to have ameans and methods that are capable of increasing the levels of thesesecondary metabolites in plants.

EP 1671534 A1 describes a method of increasing the content of depsides,preferably dicaffeoylquinic acids and/or dicaffeoyltartaric acids in aplant by treating the plant with biotic and/or abiotic stimuli.

U.S. Pat. No. 7,666,455 teaches a method for increasing the amount ofresveratrol in a peanut material by size-reducing the peanut kernel,abiotically stressing the size-reduced peanut kernel, and incubating theabiotically stressed size-reduced peanut kernel under conditions capableof increasing the amount of resveratrol in the size-reduced peanutkernel.

U.S. Pat. No. 9,227,898 describes a method for increasing stilbeneproduction, particularly resveratrol and piceatannol, in sugarcane byirradiating cut sides of sugarcane billets with Ultraviolet-C orUltraviolet-B light.

US 2004/0234657 A1 teaches the treatment of a plant with a modifiedlecithin, e.g., enzyme-modified lecithin (EML) and chemically modifiedlecithin such as acetylated lecithin (ACL) and hydroxylated lecithin(HDL), to induce expression of phenylalanine ammonia lyase, polyphenoloxidase, and peroxidase, and enhance lignin production.

Further, wounded tobacco (Hagel, & Facchini (2005) Planta 221:904-914)and potato tuber discs (Negrel, et al. (1993) J. Plant Physiol.142(5):518-524) have been shown to produce increased levels of amides offerulic acid with tyramine or octopamine, and elicitor chitosantreatment has been shown in increase coumaroyl tyramine in potato(Schmidt, et al. (1999) J. Biol. Chem. 274:4273-4280).

SUMMARY OF THE DISCLOSURE

In aspects, the disclosure provided herein describes methods forproducing a consumable product with enhanced levels of a tyraminecontaining hydroxycinnamic acid amide. In some embodiments, the methodfor producing a consumable product with enhanced levels of a tyraminecontaining hydroxycinnamic acid amide, comprising:

-   -   (a) subjecting a plant for producing a compound of Formula I

-   -   wherein    -   R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently        selected from hydrogen, deuterium, hydroxyl, halogen, cyano,        nitro, optionally substituted amino, optionally substituted        C-amido, optionally substituted N-amido, optionally substituted        ester, optionally substituted —(O)C₁₋₆alkyl, optionally        substituted —(O)C₁₋₆alkenyl, optionally substituted        —(O)C₁₋₆alkynl, optionally substituted, —(O)C₄₋₁₂cycloalkyl,        optionally substituted —(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally        substituted —(O)C₄₋₁₂heterocyclyl, optionally substituted        —(O)C₁₋₆alkylC₄₋₁₂heterocyclyl, optionally substituted        —(O)C₄₋₁₂aryl, optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl,        optionally substituted —(O)C₁₋₁₂heteroaryl, and optionally        substituted —(O)C₁₋₆alkylC₁₋₁₂heteroaryl; the dashed bond is        present or absent;    -   X is CH₂ or O;    -   Z is CHR^(a), NR^(a), or O; and    -   R^(a) is selected from hydrogen, deuterium, hydroxyl, halogen,        cyano, nitro, optionally substituted amino, optionally        substituted C-amido, optionally substituted N-amido, optionally        substituted ester, optionally substituted —(O)C₁₋₆alkyl,        optionally substituted —(O)C₁₋₆alkenyl, optionally substituted        —(O)C₁₋₆alkynl, optionally substituted, —(O)C₄₋₁₂cycloalkyl,        optionally substituted —(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally        substituted —(O)C₄₋₁₂heterocyclyl, optionally substituted        —(O)C₁₋₆alkylC₄₋₁₂heterocyclyl, optionally substituted        —(O)C₄₋₁₂aryl, optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl,        optionally substituted —(O)C₁₋₁₂heteroaryl, and optionally        substituted —(O)C₁₋₆alkylC₁₋₁₂heteroaryl,    -   the dashed bond is present or absent,    -   to at least one biotic or abiotic stress; and    -   (b) incorporating the plant or extract into a consumable        product.

In some embodiments, the method for producing a consumable product withenhanced levels of a tyramine containing hydroxycinnamic acid amide,comprising:

-   -   (a) subjecting a plant for producing a compound of Formula II

-   -   wherein    -   R¹, R², and R³ are each independently present or absent, and        when present is a substituent on one or more ring atoms (e.g.,        position 2, 3, and/or 4) and is for each ring atom independently        a hydroxy group, halo group, substituted or unsubstituted lower        alkyl group, or substituted or unsubstituted lower alkoxy group,    -   the dashed bond is present or absent,    -   to at least one biotic or abiotic stress; and    -   (b) incorporating the plant or extract into a consumable        product.

In some embodiments, the method further comprising recovering an extractfrom the plant. In some embodiments, the method further comprisingcontacting the plant with a precursor of a tyramine containinghydroxycinnamic acid amide. In some embodiments, the biotic stress isfalse germination. In some embodiments, the at least one biotic orabiotic stress is applied post-harvest. In some embodiments, the atleast one biotic or abiotic stress is applied pre-harvest. In someembodiments, the abiotic stress is selected from at least one ofhyperosmotic stress, salt, temperature stresses, aberrant nutrientconditions, mechanical shock flooding, wounding, anaerobic stress,oxidative stress, ozone, high light, heavy metals, toxic chemicals,ultrasound, ultraviolet light, elicitor chitosan treatment, modifiedlecithin treatment, or abscisic acid treatment.

In some embodiments, the optionally recovering an extract from the plantcomprises an ethanol extract. In some embodiments, the plant is selectedfrom at least one of Tribulus terrestris, Annona montana, Annonamuricata, Annona cherimola, Annona atemoya, Solanum tuberosum, Cannabissativa, Lycium barbarum, Allium sativum, Solanum lycopersicum, Capsicumannuum, Capsicum frutescens, Solanum tuberosum, Annona spp., Lyciumbarbarum, Ipomoea batatas, Zea Mays, Piper nigrum, Dysphaniaambrosioides, Hibiscus sabdariffa, Piper auritum, Solanum lycopersicum,or Allium fistulosum.

In some embodiments, the compound is selected from p-coumaroyltyramine,n-caffeoyltyramine, n-feruloyltyramine, and sinpoyltyramine. In someembodiments, the n-feruloyltyramine yield is greater than 1000 mg/kg ofthe plant. In some embodiments, the p-coumaroyltyramine yield is greaterthan 50 mg/kg of the plant. In some embodiments, the at least one bioticor abiotic stress comprises incubating the plant at about 25° C. toabout 37° C. and a pH of 6.5 to about 9.5. In some embodiments, the atleast one biotic or abiotic stress comprises incubating the plant atabout 30° C. and a pH of about 8.5.

In some embodiments, the abiotic stress is physical wounding and thecompound of Formula I is n-feruoyltyramine. In some embodiments, thephysical wounding increases n-feruloyltyramine is increased by at least9-fold. In some embodiments, the physical wounding increasesn-feruloyltyramine is increased by at least 13-fold. In someembodiments, the physical wounding increases n-feruloyltyramine isincreased by at least 33-fold. In some embodiments, the abiotic stressis ultraviolet light and the compound of Formula I isn-feruloyltyramine, n-caffeoyltyramine, and p-coumaroyltyramine. In someembodiments, the plant is exposed to ultraviolet light for about 15 toabout 30 minutes. In some embodiments, the abiotic stress is temperaturestresses and the compound of Formula I is n-feruloyltyramine,n-caffeoyltyramine, and p-coumaroyltryamine. In some embodiments, thetemperatures stress increases the production of n-feruloyltyramine,n-caffeoyltyramine, and p-coumaroyltryamine from about 25% to about 47%.

In some embodiments, a consumable product is produced by the method asdescribed herein. In some embodiments, the consumable product is adietary supplement, food ingredient, food additive, food product, feedproduct, a medical food, nutraceutical or pharmaceutical composition.

Some embodiments relate to a composition enriched for a tyraminecontaining hydroxycinnamic acid amide comprising, an extract or sourcematerial including one or more precursors of a tyramine containinghydroxycinnamic acid amide, wherein the extract or source material hasbeen contacted with an enzymatic material, wherein the enzymaticmaterial comprises one or more endogenous enzymes capable of convertingthe one or more precursors to the tyramine containing hydroxycinnamicacid amide.

In some embodiments, the enzymatic material comprises a phenylalanineammonia lyase, 4-courmarate-CoA ligase, cinnamate-4-hydroxylase,coumarate-3-hydroxylase, coumaroyl-CoA 3-hydroxylase, caffeoyl-CoAO-methyltransferase, ferulate-5-hydroxylase, caffeicacid/5-hydroxyferulic acid O-methyltransferase, tyrosine ammonia lyase,or a combination thereof. In some embodiments, the tyramine containinghydroxycinnamic acid amide is N-caffeoyltyramine, N-feruloyltyramine,5-hydroxyferuloyltyramine, p-coumaroyltyramine, cinnamoyltyramine,sinapoyltyramine, or a combination thereof. In some embodiments, thecomposition is a consumable product. In some embodiments, the consumableproduct is a dietary supplement, food ingredient, food additive, feedproduct, food product, a medical food, nutraceutical or pharmaceuticalcomposition.

In some embodiments, a method for enhancing levels of a tyraminecontaining hydroxycinnamic acid amide in an extract or source materialis provided herein. In some embodiments, the method comprises contactingan extract or source material including one or more precursors of atyramine containing hydroxycinnamic acid amide with an enzymaticmaterial, wherein the enzymatic material comprises one or moreendogenous enzymes capable of converting the one or more precursors tothe tyramine containing hydroxycinnamic acid amide, thereby enhancingthe levels of a tyramine containing hydroxycinnamic acid amide in theextract or source material. In some embodiments, the method furthercomprises contacting the extract or source material with a precursor ofa tyramine containing hydroxycinnamic acid amide. In some embodiments,the tyramine containing hydroxycinnamic acid amide is a compound ofFormula I. In some embodiments, the source materials is selected from atleast one of Tribulus terrestris, Annona montana, Annona muricata,Annona cherimola, Annona atemoya, Solanum tuberosum, Cannabis sativa,Lycium barbarum, Allium sativum, Solanum lycopersicum, Capsicum annuum,Capsicum frutescens, Solanum tuberosum, Annona spp., Lycium barbarum,Ipomoea batatas, Zea Mays, Piper nigrum, Dysphania ambrosioides,Hibiscus sabdariffa, Piper auritum, Solanum lycopersicum, or Alliumfistulosum.

This disclosure also provides a composition enriched for a tyraminecontaining hydroxycinnamic acid amide composed of an extract includingone or more precursors of a tyramine containing hydroxycinnamic acidamide, wherein said extract has been contacted with an enzymaticmaterial including one or more endogenous enzymatic activities thatconvert the one or more precursors to the tyramine containinghydroxycinnamic acid amide. In some embodiments, the enzymatic materialcomprises a phenylalanine ammonia lyase, 4-courmarate-CoA ligase,cinnamate-4-hydroxylase, coumarate-3-hydroxylase, coumaroyl-CoA3-hydroxylase, caffeoyl-CoA O-methyltransferase, ferulate-5-hydroxylase,caffeic acid/5-hydroxyferulic acid O-methyltransferase, tyrosine ammonialyase, or a combination thereof. In other embodiments, the tyraminecontaining hydroxycinnamic acid amide is N-caffeoyltyramine,N-feruloyltyramine, 5-hydroxyferuloyltyramine, p-coumaroyltyramine,cinnamoyltyramine or sinapoyltyramine. A consumable product, e.g., adietary supplement, food ingredient or additive, food product, a medicalfood, nutraceutical or pharmaceutical composition is also provided, asis a method for enhancing levels of a tyramine containinghydroxycinnamic acid amide in an extract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic pathway for the biosynthesis of tyraminecontaining hydroxycinnamic acid amides from hydroxycinnamoyl-CoA estersand tyramine. However, cofactors and co-substrates are not shown forclarity. Enzymes of the phenylpropanoid pathway are phenylalanineammonia-lyase (PAL, E.C. 4.3.1.24); cinnamate-4-hydroxylase (C4H, E.C.1.14.14.91); 4-coumaroyl-CoA ligase (4CL, E.C. 6.2.1.12);coumarate-3-hydroxylase (C3H, E.C. 1.14.13.-); coumaroyl-CoA3-hydroxylase (CCoA3H, or 5-O-(4-coumaroyl)-D-quinate 3′-monooxygenase,E.C. 1.14.14.96); caffeoyl-CoA O-methyltransferase (CCoAOMT, E.C.2.1.1.104); ferulate-5-hydroxylase (FSH, E.C. 1.14.-.-); and caffeicacid/5-hydroxyferulic acid O-methyltransferase (COMT, E.C. 2.1.1.68).Additional enzymes in the biosynthesis of tyramine containinghydroxycinnamic acid amides include hydroxycinnamoyl CoA:tyraminehydroxycinnamoyltransferase (THT, E.C. 2.3.1.110); tyrosine ammonialyase (TAL, E.C. 4.3.1.23), phenylalanine hydroxylase (PAH, E.C.1.14.16.1) and tyrosine decarboxylase (TYDC, E.C. 4.1.1.25).

FIG. 2 shows the amounts of N-trans-caffeoyltyramine,N-trans-feruloyltyramine and p-coumaroyltyramine present in ethanolextracts (% of extract, w/w) from a variety of sources includingTribulus terrestris seed (1), Cannabis (hemp) seed hull (2), Annona spp.(atemoya) seed (3), Annona muricata (Guanabana) seed (4), A. cherimola(Cherimoya) leaf (5), Zea mays stalk (6), Tribulus terrestris (GoatHead) seed (7), A. cherimola hardwood (bark and core) (8), Solanumlycopersicum ground pomace (9), S. tuberosum (yellow potato) peel (10),Piper nigrum (black peppercorn) fruit (11), S. tuberosum (purple potato)peel (12), S. tuberosum (red potato) peel (13), S. lycopersicum pomace(14), S. lycopersicum extruded pomace (15), A. muricata (Guanabana)leaves (16), Allium sativum (garlic) bulb (17), S. tuberosum (purplepotato) peel (18), A. montana (Mountain soursop) leaves (19), Z. maysleaves (20), S. tuberosum (purple potato) sprouts (21), A. cherimola(Cherimoya) seed (22), Allium fistulosum (green onion) whole plant (23),S. tuberosum (white potato) peel (24), A. cherimola (Cherimoya)greenwood (25), Cannabis (hemp) leaves (26), S. tuberosum (white potato)peel (27), S. lycopersicum seed (28), S. lycopersicum (Beefsteak) wholefruit (29), A. muricata (Guarabana) skin of unripe fruit (30), A.muricata (Guanabana) ripe fresh fruit (31), A. squamosa (sweetsop) wholefruit (32), Capsicum annuum (serrano pepper) fruit (33), S. tuberosum(Russet potato) peel (34), Lycium barbarum (goji/wolf berry) fruit (35),S. tuberosum (purple potato) core (36), Chenopodium quinoa (quinoa) seed(37), Ipomoea batatas (sweet potato) whole potato (38), Ipomoea batatas(sweet potato) peel (39), Armoracia rusticana (horseradish) root (40),S. tuberosum (Colorado potato) peel (41), Fagopyrum esculentum(buckwheat) hulls (42), Capsicum frutescens (piri pepper) fruit (43), S.tuberosum (purple potato) core (44), C. annuum (Thai chili) stems andleaves (45), A. muricata (Guanabana) unripe fruit flesh (46), S.tuberosum (yellow potato) core (47), and Eragrostis tef (teff) seed(48).

FIG. 3 shows the amounts of N-trans-feruloyltyramine andp-coumaroyltyramine present in ethanol extracts (mg compound/kg dryplant material) from additional sources including Piper nigrum(peppercorn) fruit (1), Dysphania ambrosioides (epazote) leaf (2),Hibiscus sabdariffa (hibiscus) roselle (3), and Piper auritum (hojasanta) leaf.

FIG. 4 shows the effect of wounding stress on the production ofN-trans-feruloyltyramine in the core of a Solanum tuberosum tuber 1, 5and 9 days after wounding the peeled tuber.

FIGS. 5A and 5B show the effect of radiation stress on the production ofN-trans-feruloyltyramine in peppercorns after a 15- or 30-minute UV-Cexposure (FIG. 5A) or N-trans-feruloyltyramine,N-trans-caffeoyltyramine, and p-coumaroyltyramine in graviola leaves andgreen onions after a 15-minute UV-C exposure (FIG. 5B) as compared tocontrol, unexposed plant tissue (CNTL).

FIG. 6 shows the effect of false germination of hemp seeds on tyraminecontaining hydroxycinnamic acid amide production. Toasted hemp seedswere soaked in distilled water for 5 days and sampled daily forN-trans-caffeoyltyramine (M1), N-trans-feruloyltyramine (M2), orp-coumaroyltyramine (M3).

FIG. 7 shows the effect of soaking temperature and toasting conditionson the combined enrichment of N-trans-caffeoyltyramine,N-trans-feruloyltyramine, and p-coumaroyltyramine via false germination.

FIG. 8 shows the effect of combining different stresses on theproduction of N-trans-feruloyltyramine. Red potatoes were sliced (i.e.,wounded) and dipped into aqueous solutions of 10 mg/ml liveendomycorrhizal fungi, 10 mg/ml inactivated cordy-gen fungi, or 1 mg/mllaminaran (polysaccharide from brown algae).

FIG. 9 shows the effect of combining a stress with a precursor on theproduction of feruloyltyramine. Red potatoes were sliced (i.e., andboiled in water for 6 minutes or dipped into abiotic N-trans-wounded)aqueous solutions of 400 μM tyramine or citric acid (pH ˜4).

DETAILED DESCRIPTION OF THE DISCLOSURE

Tyramine-derived N-hydroxycinnamic acid amides (HCAAs) are commonlyassociated with the cell wall of tissues near pathogen-infected or woundhealing regions of plants. Moreover, feruloyltyramine andferuloyloctapamine are covalent cell wall constituents of both naturaland wound periderms of potato (Solanum tuberosum) tubers. The depositionof HCAAs is thought to create a barrier against pathogens by reducingcell wall digestibility.

Tyramine containing hydroxycinnamic acid amides have now been shown toexhibit agonistic activity toward HNF4α (hepatocyte nuclear factor 4α),a global nuclear transcription factor that regulates expression of genesinvolved in maintaining balanced metabolism (homeostasis). By agonizingHNF4α activity, the plant-specific tyramine derivatives find use inmitigating the adverse effects of free fatty acids, modulatingmetabolism, improving digestive health and addressing the underlyingpathogenesis of metabolic disorders, such as nonalcoholic fatty liverdisease, nonalcoholic steatohepatitis and type II diabetes mellitus.

Accordingly, the present disclosure provides compositions with enhancedlevels of one or more tyramine containing hydroxycinnamic acid amides.In some embodiments, the compositions are prepared by contacting anextract including one or more precursors of a tyramine containinghydroxycinnamic acid amide with an enzymatic material including one ormore endogenous enzymatic activities that convert the one or moreprecursors to the tyramine containing hydroxycinnamic acid amide.Alternatively, or in addition to, enhanced levels of a tyraminecontaining hydroxycinnamic acid amide can be achieved by subjecting aplant to at least one biotic or abiotic stress, optionally recovering anextract from the plant; and incorporating the plant or extract into aconsumable product. The present in situ methods allow for increasedyield of tyramine containing hydroxycinnamic acid amides in plantextracts or fractions thereof thereby reducing downstream processing andpurification costs.

In some aspects, a tyramine containing hydroxycinnamic acid amide hasthe structure of Formula I and includes homodimers, heterodimers, andconjugates thereof:

In some embodiments, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are eachindependently selected from hydrogen, deuterium, hydroxyl, halogen,cyano, nitro, optionally substituted amino, optionally substitutedC-amido, optionally substituted N-amido, optionally substituted ester,optionally substituted —(O)C₁₋₆alkyl, optionally substituted—(O)C₁₋₆alkenyl, optionally substituted —(O)C₁₋₆alkynl, optionallysubstituted, —(O)C₄₋₁₂cycloalkyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally substituted—(O)C₄₋₁₂heterocyclyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂heterocyclyl, optionally substituted —(O)C₄₋₁₂aryl,optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl, optionally substituted—(O)C₁₋₁₂heteroaryl, and optionally substituted—(O)C₁₋₆alkylC₁₋₁₂heteroaryl.

In some embodiments, R¹, R², R³, and R⁸ are each independently selectedfrom hydrogen, deuterium, hydroxyl, halogen, cyano, nitro, optionallysubstituted amino, optionally substituted C-amido, optionallysubstituted N-amido, optionally substituted ester, optionallysubstituted —(O)C₁₋₆alkyl, optionally substituted —(O)C₁₋₆alkenyl,optionally substituted —(O)C₁₋₆alkynl, optionally substituted,—(O)C₄₋₁₂cycloalkyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally substituted—(O)C₄₋₁₂heterocyclyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂heterocyclyl, optionally substituted —(O)C₄₋₁₂aryl,optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl, optionally substituted—(O)C₁₋₁₂heteroaryl, and optionally substituted—(O)C₁₋₆alkylC₁₋₁₂heteroaryl, and R⁴, R⁵, R⁶, R⁷, and R⁹ are eachindependently hydrogen, deuterium, hydroxyl, or halogen;

In some embodiments, R¹, R², and R⁸ are each independently selected fromhydrogen, deuterium, hydroxyl, halogen, cyano, nitro, optionallysubstituted amino, optionally substituted C-amido, optionallysubstituted N-amido, optionally substituted ester, optionallysubstituted —(O)C₁₋₆alkyl, optionally substituted —(O)C₁₋₆alkenyl,optionally substituted —(O)C₁₋₆alkynl, optionally substituted,—(O)C₄₋₁₂cycloalkyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally substituted—(O)C₄₋₁₂heterocyclyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂heterocyclyl, optionally substituted —(O)C₄₋₁₂aryl,optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl, optionally substituted—(O)C₁₋₁₂heteroaryl, and optionally substituted—(O)C₁₋₆alkylC₁₋₁₂heteroaryl, and R³, R⁴, R⁵, R⁶, R⁷, and R⁹ are eachindependently hydrogen, deuterium, hydroxyl, or halogen.

In some embodiments, the dashed bond is present or absent.

In some embodiments, X is CH₂ or O.

In some embodiments, Z is CHR^(a), NR^(a), or O.

In some embodiments, R^(a) is selected from hydrogen, deuterium,hydroxyl, halogen, cyano, nitro, optionally substituted amino,optionally substituted C-amido, optionally substituted N-amido,optionally substituted ester, optionally substituted —(O)C₁₋₆alkyl,optionally substituted —(O)C₁₋₆alkenyl, optionally substituted—(O)C₁₋₆alkynl, optionally substituted, —(O)C₄₋₁₂cycloalkyl, optionallysubstituted —(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally substituted—(O)C₄₋₁₂heterocyclyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂heterocyclyl, optionally substituted —(O)C₄₋₁₂aryl,optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl, optionally substituted—(O)C₁₋₁₂heteroaryl, and optionally substituted—(O)C₁₆alkylC₁₋₁₂heteroaryl.

In some embodiments, a tyramine containing hydroxycinnamic acid amidehas the structure of Formula II and includes homodimers, heterodimers,and conjugates thereof (for example lignanamides),

-   -   wherein

R¹ is present or absent, and when present is a substituent on one ormore ring atoms (e.g., position 2, 3, and/or 4) and is for each ringatom independently a hydroxy group, halo group, substituted orunsubstituted lower alkyl group, or substituted or unsubstituted loweralkoxy group; R² is present or absent, and when present is a substituenton one or more ring atoms (e.g., position 2, 3, and/or 4) and is foreach ring atom independently a hydroxy group, halo group, substituted orunsubstituted lower alkyl group, or substituted or unsubstituted loweralkoxy group, R³ is present or absent, and when present is a substituenton one or more ring atoms (e.g., position 2, 3, and/or 4) and is foreach ring atom independently a hydroxy group, halo group, substituted orunsubstituted lower alkyl group, or substituted or unsubstituted loweralkoxy group, and the dashed bond is present or absent. In accordancewith this disclosure, a tyramine containing hydroxycinnamic acid amideincludes both cis and trans isomers.

For the groups herein, the following parenthetical subscripts furtherdefine the groups as follows: “(C_(n))” defines the exact number (n) ofcarbon atoms in the group. For example, “C₁-C₁₆-alkyl” designates thosealkyl groups having from 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6,or any range derivable therein (e.g., 3-6 carbon atoms)).

The term “lower alkyl” is intended to mean a branched or unbranchedsaturated monovalent hydrocarbon radical containing 1 to 6 carbon atoms(i.e., C₁-C₆-alkyl), such as methyl, ethyl, propyl, isopropyl,tert-butyl, butyl, n-hexyl and the like.

Similarly, a lower alkoxy group is a C₁-C₆-alkoxy group having thestructure —OR wherein R is “alkyl” as defined further above. Particularalkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, tert-butoxy, iso-butoxy, sec-butoxy, n-pentoxy,1,2-dimethylbutoxy, and the like.

The term “halo” is used herein to refer to chloro (Cl), fluoro (F),bromo (Br) and iodo (I) groups. In some embodiments, the halo group is afluoro group.

In any of the groups described herein, a substituted group (e.g., asubstituted lower alkyl group or substituted lower alkoxy group) refersto an available hydrogen being replaced with an alkyl, alkenyl, alkynyl,aryl, heteroaryl, aralkyl, alkylaryl, heteroaralkyl, heteroarylalkenyl,heteroarylalkynyl, alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy,aryloxy, aralkoxy, alkoxyalkoxy, acyl, halo, nitro, cyano, carboxy,aralkoxycarbonyl, heteroarylsulfonyl, alkoxycarbonyl, alkylsulfonyl,alkylthio, arylthio, aryloxycarbonyl, arylsulfonyl, heteroarylthio,aralkylthio, heteroaralkylthio, cycloalkyl, heterocyclyl or glycosylgroup.

In some embodiments, the disclosure encloses a compound of Formula(III):

In some embodiments, R¹, R², R³, and R⁴ are each independently selectedfrom hydrogen, deuterium, hydroxyl, halogen, cyano, nitro, optionallysubstituted amino, optionally substituted C-amido, optionallysubstituted N-amido, optionally substituted ester, optionallysubstituted —(O)C₁₋₆alkyl, optionally substituted —(O)C₁₋₆alkenyl,optionally substituted —(O)C₁₋₆alkynl, optionally substituted,—(O)C₄₋₁₂cycloalkyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally substituted—(O)C₄₋₁₂heterocyclyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂heterocyclyl, optionally substituted —(O)C₄₋₁₂aryl,optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl, optionally substituted—(O)C₁₋₁₂heteroaryl, and optionally substituted—(O)C₁₋₆alkylC₁₋₁₂heteroaryl.

In some embodiments, the dashed bond is present or absent.

In some embodiments, Z is CHR^(a), NR^(a), or O.

In some embodiments, R^(a) is selected from hydrogen, deuterium,hydroxyl, halogen, cyano, nitro, optionally substituted amino,optionally substituted C-amido, optionally substituted N-amido,optionally substituted ester, optionally substituted —(O)C₁₋₆alkyl,optionally substituted —(O)C₁₋₆alkenyl, optionally substituted—(O)C₁₋₆alkynl, optionally substituted, —(O)C₄₋₁₂cycloalkyl, optionallysubstituted —(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally substituted—(O)C₄₋₁₂heterocyclyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂heterocyclyl, optionally substituted —(O)C₄₋₁₂aryl,optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl, optionally substituted—(O)C₁₋₁₂heteroaryl, and optionally substituted—(O)C₁₋₆alkylC₁₋₁₂heteroaryl.

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.

In some embodiments, the tyramine containing hydroxycinnamic acid amidehas a structure of Formula IV:

wherein,

-   -   R² is present or absent, and when present is a hydroxy or        methoxy group;    -   R³ is present or absent, and when present is a hydroxyl group or        methoxy group; and    -   R⁴ is present or absent, and when present is a hydroxy or        methoxy group.

“Isomer” refers to especially optical isomers (for example essentiallypure enantiomers, essentially pure diastereomers, and mixtures thereof)as well as conformation isomers (i.e., isomers that differ only in theirangles of at least one chemical bond), position isomers (particularlytautomers), and geometric isomers (e.g., cis-trans isomers).

In some embodiments, the disclosure encloses a compound of Formula (V):

In some embodiments, R³ and R⁴ are each independently selected fromhydrogen, deuterium, hydroxyl, halogen, cyano, nitro, optionallysubstituted amino, optionally substituted C-amido, optionallysubstituted N-amido, optionally substituted ester, optionallysubstituted —(O)C₁₋₆alkyl, optionally substituted —(O)C₁₋₆alkenyl,optionally substituted —(O)C₁₋₆alkynl, optionally substituted,—(O)C₄₋₁₂cycloalkyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally substituted—(O)C₄₋₁₂heterocyclyl, optionally substituted—(O)C₁₋₆alkylC₂₋₁₂heterocyclyl, optionally substituted —(O)C₅₋₁₂aryl,optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl, optionally substituted—(O)C₁₋₁₂heteroaryl, and optionally substituted—(O)C₁₋₆alkylC₁₋₁₂heteroaryl.

In some embodiments, the each independently selected dashed bond ispresent or absent.

In some embodiments, Z is CHR^(a), NR^(a), or O.

In some embodiments, R^(a) is selected from hydrogen, deuterium,hydroxyl, halogen, cyano, nitro, optionally substituted amino,optionally substituted C-amido, optionally substituted N-amido,optionally substituted ester, optionally substituted —(O)C₁₋₆alkyl,optionally substituted —(O)C₁₋₆alkenyl, optionally substituted—(O)C₁₋₆alkynl, optionally substituted —(O)C₄₋₁₂cycloalkyl, optionallysubstituted —(O)C₄₋₁₂heterocyclyl, optionally substituted—(O)C₄₋₁₂cycloalkyl, optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl,optionally substituted —(O)C₁₋₆alkylC₅₋₁₂heteroaryl.

In some embodiments, Q^(a), Q^(b), Q^(c), Q^(d) are each independentlyselected from a bond, CHR^(a), NR^(a), C═O, and —O—.

In some embodiments, R^(a) is selected from hydrogen, deuterium,hydroxyl, halogen, cyano, nitro, optionally substituted amino,optionally substituted C-amido, optionally substituted N-amido,optionally substituted ester, optionally substituted —(O)C₁₋₆alkyl,optionally substituted —(O)C₁₋₆alkenyl, optionally substituted—(O)C₁₋₆alkynl, optionally substituted, —(O)C₄₋₁₂cycloalkyl, optionallysubstituted —(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally substituted—(O)C₄₋₁₂heterocyclyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂heterocyclyl, optionally substituted —(O)C₄₋₁₂aryl,optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl, optionally substituted—(O)C₋₁₂heteroaryl, and optionally substituted—(O)C₁₋₆alkylC₁₋₁₂heteroaryl.

In some embodiments, Q^(c), Q^(d) are absent. In some embodiments, Q^(d)is absent.

In some embodiments, n is 1, 2, 3, or 4

In some embodiments, the compound of Formula I, II, III, and IV areselected from caffeoyltyramine, feruloyltyramine, coumaroyltyramine,cinnamoyltyramine, sinapoyltramine, and 5-hydroxyferuloyltyramine. Insome embodiments, the compound of Formula I, II, III, and IV areselected from n-caffeoyltyramine, n-feruloyltyramine,n-coumaroyltyramine, n-cinnamoyltyramine, n-sinapoyltyramine, and5-hydroxyferuloyltyramine. In some embodiments, the tyramine containinghydroxycinnamic acid amide is one of the following compounds:

The biosynthesis of hydroxycinnamic acid amides of tyramine by higherplants is via the phenylpropanoid pathway, specifically thehydroxycinnamic acid tyramine amide biosynthesis pathway, which involvescoupling of a tyramine moiety and a hydroxycinnamic acid-derived moiety.The amide coupling reaction is performed bytyramine-N-hydroxycinnamoyltransferase (formerly referred to astyramine-N-feruloyltransferase), or THT (E.C. 2.3.1.110), whichcondenses the activated Coenzyme A (CoA) form of the specifichydroxycinnamic acid derivative together with tyramine.

Tyramine and hydroxycinnamic acid moieties are both produced through theshikimic acid pathway that yields aromatic amino acids and folatecompounds. Tyrosine, the precursor of tyramine is produced fromprephenate, an intermediate in the shikimic acid pathway in the plastidof the plant. Prephenate, derived from the central shikimic acid pathwayintermediate chorismite via chorismite mutase, is converted to arogenatethrough a transaminase reaction via glutamate prephenateaminotransferase (E.C. 2.6.1.79), using glutamine as the amine donor, oraspartate prephenate aminotransferase (E.C. 2.6.1.78), using asparagineas the amine donor. Arogenate is then converted to tyrosine viaarogenate dehydratase (E.C. 4.2.1.91) or arogenate dehydrogenase (E.C.1.3.1.43). Finally, tyrosine is converted to tyramine via tyrosinedecarboxylase (E.C. 4.1.1.25).

Hydroxycinnamic acid moieties are produced through conversion ofphenylalanine, which, like tyrosine, is produced from arogenate viaarogenate dehydratase. Phenylalanine is then converted totrans-cinnamate via phenylalanine ammonia lyase (E.C. 4.3.1.24), whichcatalyzes a deamination step. Trans-cinnamate is converted to4-hydoxycinnamate via trans-cinnamate 4-monooxygenase (E.C. 1.14.14.91).4-Hydroxycinnamate and Coenzyme A are then converted to 4-coumaroyl-CoAvia 4-coumarate ligase (E.C. 6.2.1.12). Activated CoA forms of the otherhydroxycinnamate family members, including caffeic acid and ferulicacid, are derived from 4-coumaroyl-CoA.

Hydroxycinnamic acid amides of tyramine are synthesized by condensationof cinnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, andsinapoyl-CoA with tyramine via tyramine-N-hydroxycinnamoyltransferase(E.C. 2.3.1.110), also known as tyramine-N-feruloyltransferase, to yieldcinnamoyltyramine, p-coumaroyltyramine, N-caffeoyltyramine,N-feruloyltyramine, and sinapoyltyramine, respectively. A schematic ofthe biochemical pathways is provided in FIG. 1 .

While in principle any plant may be used in accordance with the presentdisclosure, tyramine containing hydroxycinnamic acid amides have beenshown to be synthesized in plants from genera including Solanum sp.(e.g., tomato, potato, nettle, chili pepper, and eggplant), Capsicum(e.g., piri piri pepper and searrano pepper), Allium sp. (e.g., garlic,onion, and leek), Tribulus sp. (e.g., puncture vine) and Annona sp.(e.g., cherimoya, custard apple and sweetsop). Of the plant speciestested, most were found to produce the compounds of interest in titersof less than 1% in an ethanol extract by weight (FIG. 2 ). Inparticular, Annona muricata (guanabana) was found to produce the highestlevels of N-trans-caffeoyltyramine and p-coumaroyltyramine, but only lowlevels of N-trans-feruloyltyramine. By comparison, Annona atemoyaproduced the second highest titer of N-trans-caffeoyltyramine and hightiters of both p-coumaroyltyramine and N-trans-feruloyltyramine.Further, red potato peels (Solanum tuberosum) contained trace quantitiesof N-trans-caffeoyltyramine, high levels of N-trans-feruloyltyramine andthe highest titer of p-coumaroyltyramine. Green onion displayed thesecond highest quantities of p-coumaroyltyramine (second to potatopeels), modest levels of N-trans-feruloyltyramine, and no detectableamount of N-trans-caffeoyltyramine.

In accordance with one aspect of the present disclosure, a plant issubjected to at least one biotic or abiotic stress or stimulus in orderto increase the content of phenolic compounds, especially tyraminecontaining hydroxycinnamic acid amides and/or substrates for theproduction thereof. The term “plant” includes whole plants; plant partssuch as shoot vegetative organs/structures (for example, leaves, stemsand tubers), roots, flowers and floral organs/structures (for example,bracts, sepals, petals, stamens, carpels, anthers and ovules), seed(including embryo, endosperm, and seed coat) and fruit (the matureovary); plant tissue (for example, vascular tissue, ground tissue, andthe like); and cells (for example, guard cells, egg cells, and thelike), and progeny and cultures or cell lines of the same.

The plant can be subjected to at least one biotic or abiotic stress orstimulus pre-and/or post-harvest and subsequently be used for thepreparation of plant-derived extracts including juices, infusions, andfermentation residues. The products fermentation plant-derived extractsor processed fractions thereof (e.g., including purified tyraminecontaining hydroxycinnamic acid amides) find use in consumablecompositions such as health-promoting compositions or tonics for humansand animals, as well as cosmetics.

At least one used to increase hydroxycinnamic acid biotic and/or levelsof amides (e.g., abiotic treatments is tyramine containingN-caffeoyltyramine, N-feruloyltyramine, p-coumaroyltyramine,cinnamoyltyramine or sinapoyltyramine) or precursors thereof in a plant.In some embodiments, more than one biotic and/or abiotic treatment isused, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10. In other embodiments, at leastone biotic or abiotic stress is applied pre-harvest and at least onebiotic or abiotic stress is applied post-harvest. For the purposes ofthis disclosure, the term “harvest” refers to the process or period intime in which a plant or plant part is removed from its naturalenvironment. For example, a whole plant is harvested when it is removedfrom the soil in which it was planted, whereas a fruit is harvested whenit is removed from the whole plant.

A “biotic” stress or stimulus is defined as a stress that occurs as aresult of damage done to an organism by other living organisms, such asbacteria, viruses, fungi, parasites, beneficial and harmful insects,weeds, and cultivated or native plants. For example, among theaccumulating phenylpropanoids, N-hydroxycinnamoyl-tyramines have beenidentified both in Phytophthora infestans-infected leaves andsuspension-cultured potato cells (Keller, et al. (1996) Phytochemistry42:389-396) The P. infestans-induced pathway in potato has also beenshown to occur in response to elicitor of Nicotiana glutinosa andtreatment in cultured cells Eschscholtzia californica (Villegas &Brodelius (1990) Physiol. Plant. 78:414-420), as well as Nicotianatabacum (Negrel & Javelle (1995) Physiol. Plant. 95:569-574). Similarly,tomato plants inoculated with Pseudomonas syringae pv. tomato have beenshown to accumulate p-coumaroyltyramine and feruloyltyramine (Zacares,et al. (2007) Mol. Plant Microbe Interact. 20(11)1439-48).

Examples of suitable biotic stimuli of use in the method of thisdisclosure include, but are not limited to, Phytophthora infestans,Pseudomonas syringae, Xanthomonas campestris pv. vesicatoria, Erwiniacarotovora subsp. Carotovora, Ralstonia solanacearum, Pseudomonascorrugata, Alternaria, Rhizoctonia, Sclerotinia, Colletotrichum sp.,Phythium sp., Verticillium, Fusarium wilt, late blight, spotted wiltvirus, tomato mosaic virus, fruitworm, root-knot nematode, Potato virusY, Tomato yellow leaf curl, Tomato mosaic, Tomato mottle, black leg,powdery mildew, powdery scab, leafroll virus, Braephratiloides cubense,and thrips. Food-grade fungi such as Aspergillus sojae may also be usedto enhance production of phenolic compounds.

An “abiotic” stress or stimulus is defined as a negative impact of anon-living factor on a living organism. Examples of abiotic stresses ofuse in the method of this disclosure include, but are not limited to,hyperosmotic stresses such as drought or high salt, temperature stressessuch as cold or heat, aberrant nutrient conditions, mechanical shock,flooding, wounding, anaerobic stress, oxidative stress, ozone, highlight, heavy metals, toxic chemicals, ultrasound, ultraviolet light,elicitor chitosan treatment, modified lecithin treatment, abscisic acidtreatment, false germination and combinations thereof.

By way of illustration, nitrogen depletion, temperature, and light havebeen shown to synergistically increase the content of phenolic compoundsand gene expression in the leaves of tomato (Lovdal, et al. (2010)Phytochemistry 71:605-613). Further, tyrosine decarboxylase and tyraminehydroxycinnamoyl transferase levels are increased in wounded tobacco(Hagel, & Facchini (2005) Planta 221:904-914) and potato tuber discs(Negrel, et al. (1993) J. Plant Physiol. 142(5):518-524) with concurrentin vivo production of amides of ferulic acid with tyramine oroctopamine. Moreover, elicitor chitosan treatment has been shown inincrease coumaroyl tyramine in potato (Schmidt, et al. (1999) J. Biol.Chem. 274:4273-4280).

Hyperosmotic stresses include exposure to drought, high salt or highsolute conditions. Whereas drought can be achieved by reducing oreliminating the amount of water a plant receives, a high salt orhyperosmotic condition can include exposing a plant to a solutioncontaining, e.g., at least 150 mM NaCl or at least 300 mM mannitol. Aplant can be flooded or water logged by covering or submerging the plantin water.

Temperature stress includes exposure to either high or low temperature.Low temperature or freezing stress may be conditions in which theaverage temperature of the plant environment is 15° C. or lower, andstill more severely 5° C. more severely 10° C. or lower, or lower. Hightemperature stress may be conditions in which an average temperature ofthe plant environment is 25° C. or higher, more severely 30° or higher,and still more severely 35° C. or higher.

Aberrant nutrient conditions refer to high or low nitrogen, phosphorus,iron and the like.

The term “anaerobic stress” means any reduction in oxygen levelssufficient to produce a stress as hereinbefore defined, includinghypoxia and anoxia.

Oxidative stress refers to any stress, which increases the intracellularlevel of reactive oxygen species.

Wounding is the irreversible disturbance of the natural plant, tissueand/or cell structure by methods like cutting, slicing, abrasion,squashing, breaking, peeling, crushing, pressing, slashing, grinding,fluid injection, osmotic shock, detaching, shredding, rubbing, piercing,pinching and tearing.

Ultraviolet light has been reported to be an abiotic stress that inducesan increase in phenolic compounds. UVB has been the most frequently usedsource of irradiation for increasing phenol antioxidant production inplants. The UVB spectral band (280-315 μm) contributes less than 2% ofthe short-wave photons in sunlight. Post-harvest application of UVBirradiation at light ranges from about 10 mW/cm² to about 50 mW/cm² canbe carried out by known methods (Huyskens-Keil (2007) J. Appl. Bot. FoodQual. 81:140-144; Eichholz (2011) Food Chem. 126:60-64) UVC treatment(100-280 nm) at light ranges from about 1 mW/cm² to about 25 mW/cm² canalso be used to induce production of phenolics in accordance with knownmethods (Cantos, et al. (2000) J. Agric. Food Chem. 48:4606-4612).Irradiation durations depend on the UV intensity and in certainembodiments will range from about 10 minutes to about 3 hours, with somedurations between 30 minutes and 1 hour. The durations and intensitiescan be determined using routine skill in the art and will vary dependingon the commercial set up for handling large quantities of plantmaterial. In certain embodiments, irradiation is conducted attemperatures ranging from about 20-40° C.

False germination or false malting treatment similar or identical tomalting describes a technique as practiced by a person skilled in theart. However, as the seeds are in dormancy, for example in secondarydormancy, the seeds subjected to false malting do not germinate. SeeU.S. Pat. No. 10,334,689 B2, incorporated herein by reference.

The step of applying biotic or abiotic stress to a plant induces theexpression of key enzymes and/or increases pools of enzyme substrates,which in turn leads to formation and accumulation of the desiredcompound or class of compounds of Formula I. Indeed, as the dataprovided herein demonstrates, wounding of Solanum tuberosum tubers wasfound to provide a 33-fold increase in N-trans-caffeoyltyramineproduction.

In accordance with another aspect of this disclosure, the level of oneor more tyramine containing hydroxycinnamic acid amides in a plant orextract are enhanced by contacting an extract or source plant materialincluding one or more precursors of the tyramine containinghydroxycinnamic acid amides with an enzymatic material including one ormore endogenous enzymes that convert the one or precursors to one ormore tyramine containing hydroxycinnamic acid amides. In certainembodiments, the extract or source material and enzymatic material isobtained from different sources, e.g., two or more different tissues ofthe same plant, tissues from two or more different plants, or a plantand a microbe. In some embodiments, a source material including one ormore precursors of the tyramine containing hydroxycinnamic acid amidesis contacted with a source material containing one or more endogenousenzymes that convert the one or more precursors to one or more tyraminecontaining hydroxycinnamic acid amides. In another embodiment, a sourcematerial including one or more precursors of the tyramine containinghydroxycinnamic acid amides is contacted with an extract containing oneor more endogenous enzymes that convert the one or more precursors toone or more tyramine containing hydroxycinnamic acid amides. In afurther embodiment, an extract including one or more precursors of thetyramine containing hydroxycinnamic acid amides is contacted with anextract containing one or more endogenous enzymes that convert the oneor more precursors to one or more tyramine containing hydroxycinnamicacid amides.

An “extract” refers a composition containing a desired compound ofinterest which is separated from other substances present in the naturalsource material from which the composition was obtained. In someembodiments, the natural source material is a plant, microbe or animal.In certain embodiments, the extract is a bacterial or fungal extract. Inother embodiments, the extract is a plant extract. Plant extracts can beobtained from any plant tissue including a whole plant; plant part suchas shoot vegetative organs/structures (for example, leaves, stems andtubers), roots, flowers and floral organs/structures (for example,bracts, sepals, petals, stamens, carpels, anthers and ovules), seed(including embryo, endosperm, and seed coat) and fruit (the matureovary); plant tissue (for example, vascular tissue, ground tissue, andthe like); or cell (for example, guard cells, egg cells, and the like),and progeny and cultures or cell lines of the same. In some embodiments,the extract is generally recognized as safe for human consumption.Accordingly, in certain embodiments the extract is from an ediblesource. In this respect, the extract is an edible extract.

Extracts can be prepared by freezing, grinding, macerating, pulverizingand/or fermenting the source material of interest, subjecting the sourcematerial to solvent extraction, and separating the insoluble materialfrom soluble material. In this respect, an “extract” of the disclosurecan be crude, fractionated, sub-fractionated, separated, isolated,enriched or purified, without being limited thereto. The term “crude”means compounds or molecules that have not been entirely separated fromthe components of the original composition in which it was present. Inembodiments pertaining to fractions or sub-fractions, a molecule incrude extract may be subjected to partial separation to provide a lesscrude extract containing other substances. By comparison, the term“isolated” means that a compound or molecule is substantially enrichedor purified with respect to the complex cellular milieu in which itnaturally occurs, such as in a crude extract. When an isolated moleculeis enriched or purified, the absolute level of purity is not criticaland those skilled in the art can readily determine appropriate levels ofpurity according to the use to which the material is to be put. In somecircumstances, the isolated molecule forms part of a composition (forexample a more or less crude extract containing many other substances),which may for example contain other components. In other circumstances,the isolated molecule may be purified to essential homogeneity, forexample as determined spectrophotometrically, by NMR or bychromatography (for example LC-MS).

Suitable solvents for preparing an extract include, e.g., n-pentane,hexane, butane, chloroform, dichloromethane, di-ethyl ether,acetonitrile, water, butanol, isopropanol, ethanol, methanol, glacialacetic acid, acetone, norflurane (HFA134a), ethyl acetate, dimethylsulfoxide, heptafluoropropane (HFA227), and subcritical or supercriticalfluids such as liquid carbon dioxide and water, or a combination thereofin any proportion. When solvents such as those listed above are used,the resultant extract typically contains non-specific lipid-solublematerial. This can be removed by a variety of processes including“winterization”, which involves chilling to a specified temperature,typically −20° C. followed by filtration or centrifugation to removewaxy ballast, extraction with subcritical or supercritical carbondioxide or non-polar solvents (e.g., hexane) and by distillation.

An “extract including one or more precursors of the tyramine containinghydroxycinnamic acid amides” refers to an extract including one or moreof the precursors shown in FIG. 1 , i.e., tyrosine, phenylalanine,tyramine, cinnamoyl-CoA, cinnamate, p-courmaric acid, p-coumaroyl-CoA,caffeic acid, caffeoyl-CoA, ferulic acid, feruloyl-CoA, sinapic acidand/or sinapoyl-CoA. Any natural source of these precursors can be usedto provide an extract in accordance with this disclosure. By way ofillustration, natural sources of p-coumaric acid include, but are notlimited to, peanuts, navy beans, tomatoes, carrots, basil, garlic andbarley. Further, natural sources of caffeic acid include, but are notlimited to, coffee, turmeric, basil, thyme, oregano, sage, cabbage,apples, strawberries, cauliflower, radishes, green onion, mushrooms,kale and pears. Moreover, natural sources of ferulic acid includepopcorn, tomato, garlic, navy bean, bamboo shoots, and cooked sweetcorn.In addition, hydroxycinnamic acid-rich sources include, amongst others,grains, cereals, fruits, vegetables and herbs. Cheese is also a sourceof one or more precursors.

An “enzymatic material including one or more endogenous enzymes thatconvert the one or more precursors to one or more tyramine containinghydroxycinnamic acid amides” refers to an fraction, sub-fraction,extract (as described herein), isolated enzyme, enzyme complex,bacterial cell, fungal cell, plant cell, or plant tissue culture thatendogenously includes (e.g., expresses) one or more of the enzymes shownin FIG. 1 , i.e., PAL, C4H, 4CL, C3H, CCoA3H, CCoAOMT, FSH, COMT, THT,TAL, PAH, and/or TYDC. While in principle any plant may be used tosupply the enzymatic material of the present disclosure, hydroxycinnamicacid amides of tyramine have been shown to be synthesized in plants fromgenera including Solanum sp. (e.g., tomato, potato, nettle, chilipepper, and eggplant), Allium sp. (e.g., garlic, onion, and leek),Tribulus sp. (e.g., puncture vine) and Annona sp. (e.g., cherimoya,custard apple and sweetsop) Of the plant species tested, most were foundto produce the compounds of interest in titers of less than 1% in anethanol extract by weight (FIG. 2 ). In particular, Annona murica to(guanabana) was found to produce the highest levels ofN-trans-caffeoyltyramine and p-coumaroyltyramine, but only low levels ofN-trans-feruloyltyramine. By comparison, Annona atemoya produced thesecond highest titer of N-trans-caffeoyltyramine and high titers of bothp-coumaroyltyramine and N-trans-feruloyltyramine. Further, red potatopeels (Solanum tuberosum) contained trace quantities ofN-trans-caffeoyltyramine, high levels of N-trans-feruloyltyramine andthe highest titer of p-coumaroyltyramine. Green onion displayed thesecond highest quantities of p-coumaroyltyramine (second to potatopeels), modest levels of N-trans-feruloyltyramine, and no detectableamount of N-trans-caffeoyltyramine.

Moreover, tyramine is also produced by microbial-catalyzeddecarboxylation of tyrosine. Various fermentative microorganisms,especially the lactic acid bacteria, express the tdcA gene, whichencodes for the tyrosine decarboxylase enzyme. An example of thisactivity is found in the bioconversion of tyrosine to tyramine by theEnterococcus durans which is found in cheese products.

To produce the desired tyramine containing hydroxycinnamic acid amidesof this disclosure, certain embodiments include contacting the extractor source material with the enzymatic material. In the context of thisdisclosure, “contacted” or “contacting” refers to the bringing togetherof the extract or source material and enzymatic material to facilitatethe conversion of precursors to one or more tyramine containinghydroxycinnamic acid amides. In some embodiments, contact can beachieved by passing the extract over a solid surface with the enzymaticmaterial bound thereto. In other embodiments, contact can be achieved bymixing the extract or source material with a microbe that expresses oneor more endogenous enzymes that convert the one or more precursors toone or more tyramine containing hydroxycinnamic acid amides. In certainembodiments, the mixing of an extract or source material with a microbethat expresses one or more endogenous enzymes that convert the one ormore precursors to one or more tyramine containing hydroxycinnamic acidamides further includes supplementing the mixture with tyrosine. In afurther embodiment, contact can be achieved by mixing the extract orsource material with a second extract, e.g., a plant extract, or sourcematerial that includes one or more endogenous enzymes that convert theone or more precursors to one or more tyramine containinghydroxycinnamic acid amides. Ideally, contact of the extract or sourcematerial with the enzymatic material yields an enhanced level or one ormore tyramine containing hydroxycinnamic acid amides compared to thesame extract or source material not contacted with the enzymaticmaterial. In embodiments wherein it is desirable to regulate productionof the tyramine containing hydroxycinnamic acid amides, enzymaticactivity can be enhanced by the inclusion of cofactors, modulated by pHor temperature, and/or stopped by subjecting the enzyme to an enzymedeactivation step, e.g., heat treatment.

In some embodiments, contact of a source material or extract with aprecursor of a tyramine containing hydroxycinnamic acid amide canfurther enhance the production of a tyramine containing hydroxycinnamicacid amide. Accordingly, in some embodiments, the methods of thisdisclosure further provide for the contacting a plant, source materialor extract with a precursor of a tyramine containing hydroxycinnamicacid amide including, but not limited to, tyrosine, phenylalanine,tyramine, cinnamoyl-CoA, cinnamate, p-courmaric acid, p-coumaroyl-CoA,caffeic acid, caffeoyl-CoA, ferulic acid, feruloyl-CoA, sinapic acidand/or sinapoyl-CoA.

Extracts enriched for a tyramine containing hydroxycinnamic acid amidemay be use as is or further processed by precipitation, treatment withactivated charcoal, evaporation, filtration, chromatographicfractionation, or a combination thereof. Extracts enriched for atyramine containing hydroxycinnamic acid amide are ideally obtained bychromatographic fractionation. Chromatographic fractionation typicallyincludes column chromatography and may be based on molecular sizing,charge, solubility and/or polarity. Depending on the type ofchromatographic method, column chromatography can be carried out withmatrix materials composed of, for example, dextran, agarose,polyacrylamide or silica and can include solvents such as dimethylsulfoxide, pyridine, water, dimethylformamide, methanol, saline,ethylene dichloride, chloroform, propanol, ethanol, isobutanol,formamide, methylene dichloride, butanol, acetonitrile, isopropanol,tetrahydrofuran, dioxane, chloroform/dichloromethane, etc.

As an alternative, or in conjunction with chromatography,crystallization may be performed to obtain high purity tyraminecontaining hydroxycinnamic acid amides. The solubility of the tyraminecontaining hydroxycinnamic acid amide is adjusted by changingtemperature and/or the composition of the solution, for instance byremoving ethanol, and/or adjusting the pH to facilitate precipitation,followed by filtration or centrifugation of the precipitated crystals oroils.

Typically, the product of the chromatographic step is collected inmultiple fractions, which may then be tested for the presence of thedesired compound using any suitable analytical technique (e.g., high ormedium pressure chromatography, mass spectrometry). Fractions enrichedin the desired compound may then be selected for further purification.

By way of illustration, an extract containing N-trans-caffeoyltyramineis obtained by grinding or pulverizing the plant material, subjectingthe plant material to 80% ethanol at room temperature, filtering andconcentrating the 80% ethanol extract, resuspending the concentratedextract in water, partitioning the aqueous solution with hexane, addingchloroform to the aqueous layer, and subjecting the chloroform layer toliquid chromatography with silica gel. See, e.g., Ko, et al. (2015)Internatl. J. Mol. Med. 36(4):1042-8.

An extract containing a tyramine containing hydroxycinnamic acid amidecan be standardized using conventional techniques such ashigh-performance liquid chromatography (HPLC) or high-performancethin-layer chromatography (HPTLC) The term “standardized extract” refersto an extract which is standardized by identifying characteristicingredient(s) or bioactive marker(s) present in the extract.Characterization can be, for example, by analysis of the spectral datasuch as mass spectrum (MS), infrared (IR) and nuclear magnetic resonance(NMR) spectroscopic data.

A substantially pure tyramine containing hydroxycinnamic acid amide,extract containing a tyramine containing hydroxycinnamic acid amide orplant material with enhanced levels of a tyramine containinghydroxycinnamic acid amide can be incorporated into a consumable productfor consumption by or administration to a subject. Suitable consumableproducts include, but are not limited to, a dietary supplement, foodingredient or additive, food product (e.g., a functional food), amedical food, nutraceutical or pharmaceutical composition.

Using the compositions and methods described herein, novel preparedfoods and beverages enriched in tyramine containing hydroxycinnamic acidamides can be prepared, which promote good metabolic health.Accordingly, this disclosure also provides a consumable product preparedwith the tyramine containing hydroxycinnamic acid amide-enriched extractor plant material. Examples of consumable products include, but are notlimited to, a dietary supplement, food ingredient or additive, foodproduct (e.g., a functional food), a medical food, nutraceutical orpharmaceutical composition.

A food ingredient or additive is an edible substance intended to result,directly or indirectly, in its becoming a component or otherwiseaffecting the characteristic of any food (including any substanceintended for use in producing, manufacturing, packing, processing,preparing, treating, packaging, transporting, or holding food). A foodproduct, in particular a functional food, is a food fortified orenriched during processing to include additional complementary nutrientsand/or beneficial ingredients. A food product according to thisdisclosure can, e.g., be in the form of butter, margarine, sweet orsavory spreads, biscuits, health bar, bread, cake, cereal, candy,confectionery, yogurt or a fermented milk product, juice-based andvegetable-based beverages, shakes, flavored waters, fermented beverage(e.g., Kombucha or fermented yerba mate), convenience snack such asbaked or fried vegetable chips or other extruded snack products, or anyother suitable food.

A dietary supplement is a product taken by mouth that contains acompound or extract of the disclosure and is intended to supplement thediet. A nutraceutical is a product derived from a food source thatprovides extra health benefits, in addition to the basic nutritionalvalue found in the food. A pharmaceutical composition is defined as anycomponent of a drug product intended to furnish pharmacological activityor other direct effect in the diagnosis, cure, mitigation, treatment, orprevention of disease, or to affect the structure or any function of thebody of humans or other animals. Dietary supplements, nutraceuticals andpharmaceutical compositions can be found in many forms such as tablets,coated tablets, pills, capsules, pellets, granules, softgels, gelcaps,liquids, powders, emulsions, suspensions, elixirs, syrup, and any otherform suitable for use.

In some embodiments, the enriched extract comprising a hydroxycinnamicacid amide of tyramine is combined with a carrier. The phrase “carrier”as used herein means a material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier should be compatible withthe other ingredients of the formulation and not injurious to thesubject. Some examples of materials that can serve as carriers include:(1) sugars, such as lactose, glucose and sucrose; (2) starches, such ascorn starch and potato starch; (3) cellulose, and its derivatives, suchas sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate,and hydroxyl propyl methyl cellulose; (4) powdered tragacanth; (5) malt;(6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) pH buffered solutions; (21) polyesters,polycarbonates and/or polyanhydrides; (22) surfactants, like lecithin;and (22) other non-toxic compatible substances employed in conventionalformulations.

For preparing solid compositions such as tablets or capsules, theenriched extract is mixed with a carrier (e.g., conventional tabletingingredients such as corn starch, lactose, sucrose, sorbitol, talc,stearic acid, magnesium stearate, dicalcium phosphate or gums) and otherdiluents (e.g., water) to form a solid composition. This solidcomposition is then subdivided into unit dosage forms containing aneffective amount of the compound of the present disclosure. The tabletsor pills containing the compound or extract can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction.

The liquid forms in which the compound or extract of the disclosureadministration is incorporated for oral or parenteral include aqueoussolution, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils as well as elixirs and similarvehicles. Suitable dispersing or suspending agents for aqueoussuspensions include synthetic natural gums, such as tragacanth, acacia,alginate, dextran, sodium carboxymethyl cellulose, methylcellulose,polyvinylpyrrolidone or gelatin. Liquid preparations for oraladministration may take the form of, for example, solutions, syrups orsuspensions, or they may be presented as a dry product forreconstitution with water or other suitable vehicles before use. Suchliquid preparations may be prepared by conventional means withacceptable additives such as suspending agents (e.g., sorbitol syrup,methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); preservatives (e.g., methyl or propylp-hydroxybenzoates or sorbic acid); and artificial or natural colorsand/or sweeteners.

Methods of preparing single dose formulations or compositions of thisdisclosure include the step of bringing into association an enrichedextract of the present disclosure with the carrier and, optionally, oneor more accessory and/or active ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation an enriched extract of the present disclosure with liquidcarriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product. As such, the disclosed formulation mayconsist of, or consist essentially of an enriched extract describedherein in combination with a suitable carrier.

When an enriched extract of the present disclosure is administered inthe form of a pharmaceutical, nutraceutical, or dietary supplement tohumans and animals, they can be given as a composition containing, forexample, 0.1 to 99% of active ingredient in combination with anacceptable carrier. In some embodiments, the composition includes about10% to about 30% of active ingredient in combination with an acceptablecarrier.

A consumable product may be consumed by a subject to provide less than100 mg of a compound disclosed herein per day. In certain embodiments,the consumable provides between 5 and 60 mg/day of a hydroxycinnamicacid amide of tyramine. The effective amount can be established bymethods known in the art studies and be dependent upon bioavailability,toxicity, etc.

While it is contemplated that a consumable product contains more thanone hydroxycinnamic acid amides, it is also contemplated that aconsumable product includes only an individual hydroxycinnamic acidamides. It is also contemplated that one or more extracts could becombined in any relative amounts to produce custom combinations ofingredients containing two or more tyramine containing hydroxycinnamicacid amides in desired ratios to enhance product efficacy, improveorganoleptic properties or some other measure of quality important tothe ultimate use of the product.

The method of this disclosure advantageously provides for lower-costproduction given the ability to produce a higher titer ofhydroxycinnamic acid amides of tyramine than is naturally present incertain higher plant species, thereby reducing downstream processing andpurification costs. In addition, using the method of this disclosure, itwill be possible to produce tailored, more effective compositions thanmay be possible given the compositions found in certain higher plantspecies. In effect, a composition can be produced with customizedcombinations of compounds of Formula I. Additionally, plants can betreated during processing to encourage greater activity of, e.g., THT,as well as to encourage production of greater pools of substrates.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present disclosure.

Example 1: Sources of Tyramine Containing Hydroxycinnamic Acid Amides

Ethanolic extracts were prepared from various plant species and planttissues thereof. Individual compounds were identified in the extracts byextracting dry plant powder material with 95% aqueous ethanol. Theethanol extract was concentrated and adsorbed onto CELITE® (diatomaceousearth) and dry loaded onto a C18 solid phase extraction column. Theextract was desalted by washing with two column volumes of water whichwere collected and discarded. Compounds were eluted with two columnvolumes of methanol and the extract was concentrated to dryness. Theextract was resuspended in 1:1 acetonitrile:water prior to analysis.Synthetic standards of known concentrations were used to generatecalibration curves prior to analysis. The results of this analysis arepresented in Table 1.

TABLE 1 Genus species Plant Tissue(s) N-Trans-caffeoyltyramine Tribulusterrestris seed, fruit Annona montana leaf Annona muricata peel, pulp,seed Annona cherimola pulp, seed Annona atemoya seed Solanum tuberosumpeel, tuber Cannabis sativa seed hull, leaf Lycium barbarum stemN-Trans-feruloyltyramine Allium sativum bulb Solanum lycopersicum fruitCapsicum annuum fruit Capsicum frutescens fruit Solanum tuberosum peelAnnona spp. seed, leaf, fruit Cannabis sativa seed hull, leaf Lyciumbarbarum stem, fruit Ipomoea batatas tuber Zea Mays leaf, aerial plantPiper nigrum fruit Dysphania ambrosioides leaf Hibiscus sabdariffaflower Piper auritum leaf Coumaroyltyramine Solanum lycopersicum fruitAllium fistulosum whole plant Annona spp. seed, leaf, fruit Alliumsativum bulb Annona atemoya seed Annona montana leaf Annona cherimolaseed, leaf, fruit Annona muricata seed, leaf Cannabis sativa seed hull,leaf Solanum tuberosum peel, tuber Tribulus terrestris seed, fruit Zeamays leaf, aerial plant Dysphania ambrosioides leaf Piper auritum leaf

The amounts of N-trans-caffeoyltyramine, N-trans-feruloyltyramine andp-coumaroyltyramine present in certain ethanol extracts (% of extract,w/w) was determined. Quantification of the compounds was performed bynormalizing the results by the weight of the ethanol extracts. Theresults of these analyses are presented in FIG. 2 .

Subsequent analysis of additional plant tissues identified additionalplant sources of N-trans-feruloytyramine and p-coumaroyltyramine(included in Table 1). This secondary analysis was performed similarlyto the first, but employed a different, more sensitive analyticalinstrument that allowed for direct analysis of the C18 methanol eluent,without the concentration step. Those results are presented in FIG. 3 .The mass extracted of each compound (in mg) is normalized by the mass ofdry plant material used for extraction (in kg).

Example 2: Extracts with Enhanced Levels of Tyramine ContainingHydroxycinnamic Acid Amides

Initial analytical characterization of higher plant sources of tyraminecontaining hydroxycinnamic acid amides indicate widely variable levelsof these compounds (FIG. 2 ). It is known that metabolic branch pointsthat catalyze biochemical reactions that commit substrates to specificdownstream metabolic pathways often are rate-determined steps in theoverall biochemical synthesis of a product of interest. It is positedthat the THT step is the rate-determining step in the biosynthesis oftyramine containing hydroxycinnamic acid amides. Furthermore, it isbelieved that the rate of this reaction is first substrate-limited andsecond enzyme-limited.

Therefore, to generate extracts with higher levels of tyraminecontaining hydroxycinnamic acid amides, a plant source enriched in thesubstrates of interest, specifically the hydroxycinnamic derivative orderivatives or interest, and/or tyramine-rich source, is contacted witha plant tissue source containing the THT enzyme. Plant tissue sources ofthe THT enzyme include Annona sp., A. montana (mountain soursop), A.muricata and A. cherimola, Tribulus terrestris, Allium sp., A. sativa(garlic) and A. fistulosum (green onion), Solanum lycopersicum (tomato),Capsicum sp., C. annuum (Serrano pepper), and C. frutescens (Piri Piripepper).

The THT-containing plant source and hydroxycinnamic acid-derivedsubstrate-containing plant sources are incubated under conditionssimilar to the temperature and pH conditions required for optimalactivity of the THT enzyme, i.e., 30° C. and a pH of 8.5. Moregenerally, the incubation is performed using a temperature of 25-37° C.and a pH in the range of 6.5-9.5. By way of illustration, a finishedfood or beverage product enriched in N-trans-caffeoyltyramine isgenerated by incubating the THT-containing plant tissue with a caffeoylCoA source such as Tribulus terrestris, Annona montana, Annona muricataor Annona cherimola at 30° C. and a pH of 8.5 for 1 hour. Similarly, afinished food or beverage product enriched in N-trans-feruloyltyramine,N-trans-coumaroyltyramine, or N-trans-cinnamoyltyramine THT-containingplant tissue with ferulic acid-rich, acid-rich or cinnamic acid richsources, respectively.

Fermented beverage or food products can also be produced usingmicroorganisms, including lactic acid bacteria, that produce tyramine,along with sources of one or more hydroxycinnamic acids, tyrosine andthe THT enzyme.

Example 3: Wounding Induces Production of N-Trans-Feruloyltyramine

Initial analytical characterization of higher plant sources of atyramine containing hydroxycinnamic acid amides indicate variablelevels. It is known that metabolic branch points that catalyzebiochemical reactions that commit substrates to specific downstreammetabolic pathways often are rate-determined steps in the overallbiochemical synthesis of a product of interest. It was posited that theTHT step is rate-determining in the biosynthesis of a tyraminecontaining hydroxycinnamic acid amides. Furthermore, it was believedthat that the rate of this reaction is first substrate-limited andsecond enzyme-limited. Thus, it was determined whether elevated levelstyramine containing hydroxycinnamic acid amides could be generated insitu by subjecting a plant to an abiotic stress, e.g., wounding. Inparticular, yellow tubers of Solanum tuberosum were wounded by woundingand production of N-trans-feruloyltyramine in the tuber was assessed 1,5 and 9 days after wounding. The results of this analysis indicate thatphysical wounding of the plant tissues results in a 33-fold increase inN-trans-feruloyltyramine production (FIG. 4 ). Similar to yellow potatotubers, wounding of white russet, gold and purple potato peels resultedin a 178-fold, 13-fold, and 9-fold increase in N-trans-feruloyltyramineproduction over a 14-day period post-treatment. Further analysisindicated that oxygen was required, i.e., the potatoes could not besubmerged, that citric acid/NaCl had no detrimental effect on thewounding response, and that supplying additional substrate (tyramine)enhanced the wounding response.

Example 4: UV-C Radiation Induces Production ofN-Trans-Caffeoyltyramine, N-Trans-Feruloyltyramine, andp-Coumaroyltyramine

The effect of radiation on levels of tyramine containing hydroxycinnamicacid amides was determined. In particular, peppercorns were exposed toUV-C radiation for 15 or 30 minutes, incubated at room temperature forsix hours and subsequently assayed for N-trans-feruloyltyramineproduction. The results of this analysis showed a slight increase inN-trans-feruloyltyramine production yields after the 15-minute exposureto UV-C radiation (FIG. 5A).

Similar analysis was carried out using graviola leaves or green onions.In this analysis, fresh plant material was subjected to direct UV-Cradiation for 15 minutes and subsequently incubated in the dark (about 5hours for graviola and overnight for green onions). The results of thisanalysis indicated that UV-C exposure induced a 50-300% increase inN-trans-caffeoyltyramine, N-trans-feruloyltyramine, andp-coumaroyltyramine production (FIG. 5B).

Example 5: False Germination Induces Production ofN-Trans-Caffeoyltyramine, N-Trans-Feruloyltyramine, andp-Coumaroyltyramine

The effect of false germination on levels of tyramine containinghydroxycinnamic acid amides in hemp seeds was determined. Toasted hempseeds were soaked in distilled water overnight. The water was drained,and the seeds were rinsed in fresh distilled water 2 times per day for 5days. The seeds were maintained in a moist, dark environment over thetime course of the experiment and a sample of seeds was collected everyday. For each sample collected, the seeds were dried, cracked andtreated with hexane to remove fats. The material was subsequently milledand analyzed for N-trans-feruloyltyramine, p-coumaroyltyramine, orN-trans-caffeoyltyramine production. This analysis indicated that peakinduction of N-trans-caffeoyltyramine, N-trans-feruloyltyramine, andp-coumaroyltyramine was at day 2 (1.7-fold increase; FIG. 6 ), thoughenrichment in these compounds was observed even on Day 1, immediatelyfollowing the overnight soak.

Additional studies show that the preliminary dry toasting temperatureand the distilled water soaking temperature impact the degree ofenrichment achieved (FIG. 7 ). Increasing the soaking temperature from20 degrees Celsius to 30 degrees Celsius increases the combinedenrichment of N-trans-feruloyltyramine, N-trans-caffeoyltyramine, andp-coumaroyltyramine from 25% to 47% greater than the control. Meanwhile,re-toasting the seeds for 10 minutes at 100 degrees Celsius prior tosoaking overnight in distilled water (maintained at 30 degrees Celsius)reduces the degree of enrichment achieved to 12%, presumably becausesuch toasting conditions inactivate enzymes that play critical roles inthe false germination process.

Example 6: Combined Stresses Induce Production ofN-Trans-Feruloyltyramine

Having demonstrated that wounding could increaseN-trans-feruloyltyramine production, it was determined whether combiningwounding with THT enzyme elicitors could further enhanceN-trans-feruloyltyramine production. For this analysis, red potatoeswere sliced to a standard thickness. Subsequently, the potato sliceswere dipped into solutions of 10 mg/ml live endomycorrhizal fungi, 10mg/ml inactivated cordy-gen fungi (from Mycopia), 1 mg/ml laminaran(polysaccharide from brown algae) or water (control) The potato sliceswere loosely covered to allow for air flow and incubated for 4 or 8 daysat room temperature. The results of this analysis indicated that whereaswounding in combination with laminaran exposure substantially enhancedthe production of N-trans-feruloyltyramine, wounding in combination withcordy-gen provided some enhancement and live endomycorrhizae prohibitedN-trans-feruloyltyramine production in response to wounding (FIG. 8 ).

Example 7: Wounding and Precursor Exposure Induce Production ofN-Trans-Feruloyltyramine

Having demonstrated that wounding could increaseN-trans-feruloyltyramine production, it was determined whether combiningwounding with exposure to a precursor of N-trans-feruloyltyramine couldfurther enhance N-trans-feruloyltyramine production. For this analysis,red potatoes were sliced to a standard thickness (¼ inch; twopotatoes/time point). Subsequently, the potato slices were either boiledin water for 6 minutes, or dipped into aqueous solutions of 400 μMtyramine (as precursor) or citric acid (pH ˜4; as a preservative), orwater (control). The potato slices were loosely covered to allow for airflow and incubated for 4 days at room temperature. The results of thisanalysis indicated that the combination of wounding and tyramineexposure could enhance N-trans-feruloyltyramine production by day 4,whereas citric acid had little effect (FIG. 9 ). Notably, while thetyramine dip further increased browning of the wounded potato slices,citric acid inhibited the browning pathway without impacting theproduction of N-trans-feruloyltyramine.

Example 8: Assessing Stress-Induced Increases in Enzyme Activity

Biotic and/or abiotic stresses can be used to artificially inducegreater abundance and activity of enzymes involved in tyraminecontaining hydroxycinnamic acid amide artificially induced increasedsupplies of substrates, or both. Enzyme activity assessments in responseto biotic and/or abiotic stresses can be carried out as follows.

PAL activity is assayed in a mixture (250 μL) containing 100 mM Tris-HClbuffer pH 8.0 and enzyme extract. The reaction is initiated by theaddition of 150 μL of 200 mg mL−-¹ L-phenylalanine (final concentration6 mg mL−¹) and the production of cinnamic acid is measured over 10minutes at ΔA₂₉₀.

C4H activity is assayed in a mixture (250 μL) containing 100 mMphosphate buffer (pH 7.5), 1 mM DTT, 1 mM NADPH, and 100 μL enzymeextract. The reaction is initiated by the addition of 10 mMtrans-cinnamic acid (final concentration 1 mM) and the changes inabsorbance at 290 nm are recorded during 10 minutes.

4CL activity is assayed at room temperature using a spectrophotometricassay (Knobloch & Hahlbrock (1977) Hoffm. Arch. Biochem. Biophys.184:237-248) to measure formation of CoA esters, as previously described(Lee & Douglas (1996) Plant Physiol. 112:193-205).

THT activity is assayed using known methods (Hohlfeld, et al. (1995)Plant Physiol. 107:545-552). Determination of its activity is done byHPLC coupled with a photodiode array detection (Schmidt, et al. (1998)Planta 205:51-55).

Enzymatic activities are expressed as a function of the proteinconcentration of the extracts, which are assayed and calculated usingthe Bradford method (Bradford (1976) Anal. Biochem. 72:248-54).Enzymatic activities obtained are normalized against control samples andfold change of the normalized values are calculated.

What is claimed is:
 1. A method for producing a consumable product withenhanced levels of a tyramine containing hydroxycinnamic acid amide,comprising: (a) subjecting a plant for producing a compound of FormulaIto an enzymatic material,

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are each independentlyselected from hydrogen, deuterium, hydroxyl, halogen, cyano, nitro,optionally substituted amino, optionally substituted C-amido, optionallysubstituted N-amido, optionally substituted ester, optionallysubstituted —(O)C₁₋₆alkyl, optionally substituted —(O)C₁₋₆alkenyl,optionally substituted —(O)C₁₋₆alkynl, optionally substituted,—(O)C₄₋₁₂cycloalkyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally substituted—(O)C₄₋₁₂heterocyclyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂heterocyclyl, optionally substituted —(O)C₄₋₁₂aryl,optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl, optionally substituted—(O)C₁₋₁₂heteroaryl, and optionally substituted—(O)C₁₋₆alkylC₁₋₁₂heteroaryl; the dashed bond is present or absent; X isCH₂ or O; Z is CHR^(a), NR^(a), or O; and R^(a) is selected fromhydrogen, deuterium, hydroxyl, halogen, cyano, nitro, optionallysubstituted amino, optionally substituted C-amido, optionallysubstituted N-amido, optionally substituted ester, optionallysubstituted —(O)C₁₋₆alkyl, optionally substituted —(O)C₁₋₆alkenyl,optionally substituted —(O)C₁₋₆alkynl, optionally substituted,—(O)C₄₋₁₂cycloalkyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂cycloalkyl, optionally substituted—(O)C₄₋₁₂heterocyclyl, optionally substituted—(O)C₁₋₆alkylC₄₋₁₂heterocyclyl, optionally substituted —(O)C₄₋₁₂aryl,optionally substituted —(O)C₁₋₆alkylC₅₋₁₂aryl, optionally substituted—(O)C₁₋₁₂heteroaryl, and optionally substituted—(O)C₁₋₆alkylC₁₋₁₂heteroaryl, the dashed bond is present or absent; and(b) incorporating the plant or extract into a consumable product.
 2. Themethod of claim 1, further comprising recovering an extract from theplant.
 3. The method of claim 1, further comprising contacting the plantwith a precursor of a tyramine containing hydroxycinnamic acid amide. 4.The method of claim 1, wherein the plant is selected from at least oneof Tribulus terrestris, Annona montana, Annona muricata, Annonacherimola, Annona atemoya, Solanum tuberosum, Cannabis sativa, Lyciumbarbarum, Allium sativum, Solanum lycopersicum, Capsicum annuum,Capsicum frutescens, Solanum tuberosum, Annona spp. Lycium barbarum,Ipomoea batatas, Zea Mays, Piper nigrum, Dysphania ambrosioides,Hibiscus sabdariffa, Piper auritum, Solanum lycopersicum, or Alliumfistulosum.
 5. The method of claim 1, wherein the enzymatic material isobtained from a different source than the plant.
 6. The method of claim1, wherein the enzymatic material is obtained from a microbe.
 7. Themethod of claim 1, wherein the enzymatic material is obtained from thesame source as the plant.
 8. The method of claim 1, wherein theenzymatic material comprises one or more endogenous enzymes capable ofconverting the one or more precursors to the tyramine containinghydroxycinnamic acid amide.
 9. The method of claim 1, wherein theenzymatic material comprises a phenylalanine ammonia lyase,4-courmarate-CoA ligase. cinnamate-4-hydroxylase,coumarate-3-hydroxylase, coumaroyl-CoA 3-hydroxylase, caffeoyl-CoAO-methyltransferase, ferulate-5-hydroxylase, caffeicacid/5-hydroxyferulic acid O-methyltransferase, tyrosine ammonia lyase,or a combination thereof.
 10. The method of claim 2, wherein recoveringthe extract from the plant comprises an ethanol extract.
 11. The methodof claim 1, further comprising applying an abiotic stress to the plant.12. The method of claim 10, wherein the abiotic stress is selected fromat least one of hyperosmotic stress, salt, temperature stresses,aberrant nutrient conditions, mechanical shock flooding, wounding,anaerobic stress, oxidative stress, ozone, high light, heavy metals,toxic chemicals, ultrasound, ultraviolet light, elicitor chitosantreatment, modified lecithin treatment, or abscisic acid treatment. 13.The method of claim 1, further comprising applying a biotic stress tothe plant.
 14. The method of claim 12, wherein the biotic stress isfalse germination.
 15. A method for producing a consumable product withenhanced levels of a tyramine containing hydroxycinnamic acid amide,comprising: (a) subjecting a plant for producing a compound of FormulaII to an enzymatic material,

wherein R¹, R², and R³ are each independently present or absent, andwhen present are each independently a hydroxy group, halo group,substituted or unsubstituted lower alkyl group, or substituted orunsubstituted lower alkoxy group, the dashed bond is present or absent;and (b) incorporating the plant or extract into a consumable product.16. The method of claim 15, wherein the enzymatic material is obtainedfrom a different source than the plant.
 17. The method of claim 15,wherein the enzymatic material is obtained from a microbe.
 18. Themethod of claim 15, wherein the enzymatic material is obtained from thesame source as the plant.
 19. The method of claim 15, wherein theenzymatic material comprises one or more endogenous enzymes capable ofconverting the one or more precursors to the tyramine containinghydroxycinnamic acid amide.
 20. The method of claim 15, wherein theenzymatic material comprises a phenylalanine ammonia lyase,4-courmarate-CoA, ligase, cinnamate-4-hydroxylase,coumarate-3-hydroxylase. coumaroyl-CoA 3-hydroxylase, caffeoyl-CoAO-methyltransferase, ferulate-5-hydroxylase, caffeicacid/5-hydroxyferulic acid O-methyltransferase, tyrosine ammonia lyase,or a combination thereof.